This randomized crossover study reveals that a modest amount comprising 25.0 g/d of additionally consumed LPI is capable of lowering total (−5%) and LDL cholesterol concentrations (–12%) as well as the LDL:HDL cholesterol ratio (−16%) from baseline to wk 4, primarily in subjects with higher hypercholesterolemia (> 6.6 mmol/L).
The lipid-lowering activity of dietary treatments appears to be strongly dependent on the subjects’ initial cholesterol concentrations [20, 21]. A meta-analysis of 38 human studies on soy protein ascertained that the net changes in total as well as in LDL cholesterol after intervention were directly related to the total cholesterol concentration at baseline . A more recent re-evaluation by Sirtori et al.  which included a further 33 studies on soy protein confirmed this dependency. In line with this, a subgroup analysis within the present study revealed that the total and LDL cholesterol-lowering activities of LPI and MPI were restricted to subjects with higher initial total cholesterol with an average of 7.6 mmol/L (Figure 2) indicating that there is a similar dependency between cholesterol-lowering activity and baseline cholesterol concentrations for lupin protein.
As reviewed by Anderson and Konz , a 1% increase in either total or LDL cholesterol increases the risk for coronary heart disease by 2% to 3% and 1%, respectively. Thus, after a 4 wk LPI intervention the risk for cardiovascular events such as coronary heart diseases would be reduced by 10% to 15% in subjects with higher hypercholesterolemia. Due to the lack of change in subjects with moderate hypercholesterolemia, the lipid-lowering effects for the whole study population were lower. The overall changes in plasma cholesterol after 4 wk of intervention are consistent with two other studies that show a LDL cholesterol-  and a moderate total cholesterol-lowering activity [8, 9] of 35.0 g/d lupin protein consumed by hypercholesterolemic subjects over a short-term period of 4 wk or 6 wk. A recent study conducted by our workgroup in hypercholesterolemic subjects revealed a decrease in the LDL:HDL cholesterol ratio by 7% after consumption of 40.0 g/d LPI over 8 wk, whereas total and LDL cholesterol were not altered . In contrast, Belski et al.  and Hodgson et al.  did not find changes in plasma lipid concentrations in overweight or obese participants following a long-term intervention from 16 wk to twelve months with an ad libitum diet higher in protein and fiber obtained by enriching foods with lupin flour.
The present study showed a significant reduction in systolic blood pressure by 8.4 mm Hg after 8 wk of LPI intervention. Since an increase of 1 mm Hg in systolic blood pressure is expected to increase the risk for coronary heart disease by 2.4%  the observed effect in our study could reduce the risk by 20%. These results are consistent with three previous studies that found a significant decline in blood pressure following consumption of lupin protein  or lupin flour [23, 25].
Similar to the findings of Weisse et al. , LPI intervention per se only minimally changed amino acid profile (Table 6). The concentrations of methionine were decreased by 8% after 4 wk equivalent to the observed 7% decline in the study by Weisse et al. . Since LPI relative to MPI had almost a threefold higher arginine and half the lysine proportion (Table 1), at wk 4, but not at wk 8 of LPI intervention, the lysine:arginine ratio in serum was significantly lower compared to MPI (Table 6).
Evidently, there was a general decrease in the extent of physiological effects of both protein interventions from wk 4 to wk 8. This aspect may be explained by a declining compliance to the study protocol after 4 wk of intervention, which is supported by a decrease in plasma urea from wk 4 to wk 8 (Table 5). As there was an increase in energy intake (Table 3) as well as in body weight and body fat (Table 4) after 8 wk of both protein interventions compared to baseline, we can presume that the majority of subjects did not replace an iso-caloric part of their usual diet with the protein drinks. A decrease in body weight is associated with lower concentrations of triacylglyceroles, total and LDL cholesterol as well as with higher HDL cholesterol . Thus, the observed weight gain might have additionally contributed to a worsening of the lipid profile from wk 4 to wk 8.
There were no significant differences in the plasma concentrations of cholesterol and of hs-CRP or in blood pressure between LPI and MPI intervention. This lack of treatment effects is not entirely surprising since several studies attribute milk proteins, particularly several milk peptides, with beneficial physiological properties such as hypocholesterolemic, hypotensive, and anti-inflammatory activities . Furthermore, increasing evidence indicates that the substitution of protein from animal as well as plant sources at the expense of carbohydrates may beneficially affect plasma lipids , facilitates loss of body weight  and body fat , and can lower blood pressure . The mechanisms and bioactive components of lupin protein responsible for the beneficial effects in the human body have not yet been elucidated . Contrary to soy, proteins from lupin are almost free from isoflavones  and thus physiological effects can be attributed to the protein and/or its components per se. According to Rahman et al. , the low lysine:arginine ratio might be responsible for the hypocholesterolemic properties of lupin protein. As reported by Rajamohan and Kurup , a decrease in serum cholesterol in rats was caused by a globulin fraction of sesame seeds with a low lysine:arginine ratio of 0.67 comparable to the value determined for the LPI (0.38) used in the present study. However, studies on the impact of different lysine:arginine ratios on lipid metabolism are lacking. Notably, the high proportion of arginine amounting to around 10% in lupin protein should be taken into consideration with regard to the physiological impact. Recent studies indicate that arginine is capable of modulating the concentrations of lipid signaling molecules [32, 33] and the expression of genes involved in the regulation of lipid homeostasis  which might lead to changes in the concentration of cholesterol. Hurson et al.  investigated the effect of an oral supplementation with 17 g arginine over 2 wk in elderly subjects. In the arginine-supplemented group, total cholesterol significantly decreased by 10% due mainly to reduced LDL cholesterol (-10%), whilst HDL cholesterol remained constant. These observed changes in cholesterol concentrations are in accordance with the results of the present study after 4 wk of 25 g/d LPI intervention. However, in the current study, the arginine uptake of 2.5 g/d via LPI was much lower than the supplemented 17 g/d arginine in the study by Hurson et al. . Lupin protein seems to affect the expression of hepatic genes involved in lipid metabolism as previously shown in hypercholesterolemic rats [35, 36] and, further, to alter the activity of LDL receptor as shown in a human hepatoma cell line . Supporting these results, Weisse et al.  observed an increase in mRNA abundance of the sterol regulatory element-binding protein-2 and LDL receptor along with a decrease in mRNA concentrations of 3-hydroxy-3-methylglutaryl-CoA reductase in mononuclear blood cells from hypercholesterolemic subjects after 6 wk intervention with 35 g/d lupin protein. Apart from the specific amino acid profile of lupin protein, several bioactive peptides as well as entire proteins are equally capable of demonstrating favorable properties .
In the present study, we could not detect a triacylglycerole-lowering activity of LPI. Thus, the inconsistent experimental data referring to the effect of lupin protein on triacylglyceroles [7–9] indicates the necessity of an inclusion of this parameter in further human studies. Furthermore, in future studies, it may be desirable to incorporate the test proteins in usual dietary foods in order to increase the subjects’ compliance and to avoid changes in dietary composition, thereby sustain body weight and body composition over the whole study time.